Glucagon-like peptide-1 receptor agonists, the preparation and the use of the same

ABSTRACT

The present invention provides the glucagon-like peptide-1 receptor agonists. It is indicated that the agonists have good binding capability to glucagon-like peptide-1 receptor by pharmacological tests. The present invention also provides the preparation of the agonists.

FIELD OF THE INVENTION

The present invention relates to a group of glucagon-like peptide-1receptor (GLP-1R) agonists. In particularly, the present inventionrelates to a group of small molecular organic compounds of substitutedfive-membered heterocyclic ring derivatives which may be used asnon-peptide GLP-1R agonists. The compounds of the present invention maybe used as medicaments for treating the glycometabolismdisturbance-related diseases such as type II diabetes, insensitivity toinsulin and obesity etc. And, the present invention also relates to aprocess for manufacturing the said GLP-1R agonists.

BACKGROUND OF THE INVENTION

Diabetes mellitus (DM) is a common endocrine metabolic disease withheredity tendency. It is caused mainly by the absolute or relativehyposecretion of the insulin, and it causes metabolic disturbance ofsaccharide, fat, protein, and subsequently vitamin, water andelectrolyte. The manifestations include the increase of glycemia andurine glucose, and the patients have the symptoms of polydipsia,polyphagia, polyuria, dry mouth, and general weakness. The morbidityrate of diabetes mellitus is 1 to 5%, and shows a trend of graduallyincreasing. Diabetes mellitus, cancer, and cardiovascular diseases arereferred as three worldwide serious diseases. The object of treatingdiabetes mellitus is to correct the disturbance of carbohydratemetabolism, so as to eliminate the symptoms, promote restoration of thefunction of pancreatic islet, improve the insulin resistance, maintainthe better healthy condition and physical strength, and prevent andtreat various complications.

Diabetes mellitus is commonly divided into two types: Insulin DependentDiabetes Mellitus (type I, IDDM) and Non-insulin Dependent DiabetesMellitus (type II, NIDDM). Since the pathogenesis for theses two typesof diabetes mellitus are different, the medicaments for treating themare far different, which are stated respectively as follows.

Type I diabetes mellitus is caused by virus infection in hereditarilysusceptible person which produce the paradoxical reaction of the isletcells through immunomechanism, so that the pancreatic islets begin to bedamaged and even lost their function completely. About 5% of diabetesmellitus is type I. At present, the medicaments for treating type Idiabetes mellitus mainly include exogenous insulin (including humaninsulin and animal insulin), drugs having the insulin-like effect,insulin-like growth factor-1 (IGF-1), novel long-acting insulinpreparation, and Jin Qi hypoglycemic tablet, etc.

A few of type II diabetes mellitus is caused by direct impair of β-isletcells which decreases the secretion of insulin. And most of type IIdiabetes mellitus is caused by a combination of factors that may includegenetic traits, life style, environmental contributors, metabolicdisorders, obesity, and so on. In this disease state, muscular, hepaticand adipose tissues are insensitive to the insulin thereby decreasingthe intake of the glucose. Most of diabetics suffer from type IIdiabetes mellitus. At present, the medicaments for the clinicaltreatment of NIDDM mainly include sulphonylureases, biguanides, otherhypoglycemic drugs and adjuvants, etc.

The sulphonylureas hypoglycemic drugs bind to the receptors on the cellmembrane of β-islet cells to close the potassium ion channel therebyblocking flowout of potassium ion and inducing depolarization of thecell membrane, so that the calcium ion channels are opened to allow theextracellular calcium ions flow inwardly. The increase of intracellularcalcium ions concentration triggers the release of the insulin.Sulphonylureas hypoglycemic drugs can be divided into two generationsaccording to their time of coming into existence. The first generationincludes tolpropamide, and the second generation includes glibenclamide(euglucan), gliclazide (diamicron), glipizide and gliquidone etc.

Biguanide hypoglycemic drugs inhibit appetite, improve the binding ofinsulin to the receptors, promote the anaerobic glycolysis in cells,inhibit tissue respiration and inhibit hepatic gluconeogenesis. Thebiguanide hypoglycemic drugs mainly include mefformin, phenformin andbuformin.

Other hypoglycemic drugs mainly include thiazolidinedione drugs (such astroglitazone, rosiglitazone, and pioglitazone, etc), β3-adrenoceptorregulators, glucagon receptor antagonists, fatty acid metabolisminterfering agents, α-glycosidase inhibitors (such as acarbose,voglibose, miglitol), and aldose reductase inhibitors, etc.

Recently, the development of research on glycometabolism relatedendogenous peptide hormone provides a new idea for the treatment ofdiabetes mellitus. When human body intakes nutrient materials, theenteroendocrine cells release enteropeptide hormone which mainlyincludes glucagon like peptide-1 (GLP-1) and glucose-dependentinsulinotropic peptide (GIP) and regulates metabolism by affecting theinsulin generation, gastrointestinal peristalsis, and islet cellproliferation. Wherein, GLP-1 is secreted by entero-pancreatic cells,and activates the adenylate cyclase to synthesize cAMP by highlyspecifically binding to the GLP-1 receptor of β-islet cells, so as tofurther activate the protein kinase. The metabolic signal(glycometabolism) and kinase signal (binding of GLP-1) cooperate on thecell membrane level to finally cause the Ca²⁺ channel to open and thecalcium ions to flow inwardly so that further stimulates the secretionof insulin while inhibiting the generation of glucagon, thereby decreasethe postprandial blood glucose to maintained blood glucose concentrationat a constant level. Also, GLP-1 has the function of neuroregulation,and can retard gastric emptying, and inhibit appetite. All of these aregreatly beneficial for control of diabetes mellitus. Normally, GLP-1stimulates insulin secretion depending on the blood glucoseconcentration. As the blood glucose concentration decreases, the effectof GLP-1 on stimulating insulin secretion decreases. Therefore, theaction of GLP-1 on decreasing blood glucose is self-limited, and can notcause hypoglycaemia. So, for treating diabetes mellitus, the medicamentswith the GLP-1-like action are greatly desirable for the treatment ofdiabetes mellitus. GLP-1R agonists have been one researching focus ofthe international drug development organizations. At present, theresearches on GPL-1 R mainly focus on the polypeptide regulators. Forexample, AC 2993 of Amylin Corporation has applied for clinic test in US(IND). AC2993 is a 39-amino acids polypeptide and has the effect ofpromoting the secretion of insulin as GPL-1. Since the polypeptide drugsis inconvenient for oral administration and is readily to degrade,non-peptides GLP-1R regulator is the new researching direction atpresent.

DISCLOSURE OF THE INVENTION

The object of the present invention is to design a group of novel smallmolecular organic compound of substituted five-membered heterocyclicring derivative which may be used as glucagon-like peptide-1 receptor(GLP-1R) agonists, so as to prove a way for searching the leadercompounds or the drugs for the medicaments against the diabetesmellitus. Another object of the present invention is to provide aprocess for preparing these compounds.

The Glucagon-like peptide-1 receptor agonists according the presentinvention have the specific structural formula as follows:

wherein, each of Ar₁ and Ar₂ independently is phenyl or substitutedphenyl, and the substituent groups of the said substituted phenyl isone, two or three groups optionally selected from the following groups:alkyl; hydroxyl; substituted alkoxyl or alkylamino which containssubtitutent groups including halogen, alkoxyl, or hydroxyl; substitutedalkanoylxy or alkanoylamino which contains the subtitutent groupsincluding halogen, alkoxyl, or hydroxyl; C₂-C₆ alkenyl substituted withoxygen or amine, phenyl, benzyl, C₂-C₆ enoyl, C₃-C₆ cycloalkanoyl,benzoyl, substituted benzoyl which contains optional one, two, or threesubstituent groups including alkoxyl and alkylamino, benzyloyl, thenoyl,tert-butoxycarbonyl, adamantane formoxyl

and mandeloyl

alkoxyl; alkanoylamino; cycloalkoxyl; cycloalkanoylamino; amino; amide;alkoxycarbonyl; cycloalkoxycarbonyl; alkanoylxy; alkanoylamino;cycloalkanoylxy; cycloalkanoylamino; carbamido; urylene; alkanoyl;nitro; carboxyl; and aldehyde group;

X is O, S, or NH; and

Y is O or S. When Ar₁ is

wherein R₁ is any one of the following substituent groups: H; alkyl;substituted alkyl which contains substituent groups including halogen,alkoxyl, or hydroxyl; C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl;alkanoyl; substituted alkanoyl which contains substituent groupsincluding halogen, alkoxyl, or hydroxyl; C₂-C₆ enoyl; C₃-C₆cycloalkanoyl; benzoyl; tert-butoxycarbonyl; substituted benzoyl whichcontains optional one, two, or three substituent groups includingalkoxyl and alkylamino; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and when X, is O or NH, Ar₂ is

wherein R₂ is any one of the following substituent groups: H; alkyl;substituted alkyl which contains substituent groups including halogen,alkoxyl or hydroxyl; C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl;alkanoyl; substituted alkanoyl which contains substituent groupsincluding halogen, alkoxyl, or hydroxyl; C₂-C₆ enoyl; C₃-C₆cycloalkanoyl; benzoyl; tert-butoxycarbonyl; substituted benzoyl whichcontains optional one, two or three substituent groups including alkoxyland alkylamino; benzyloyl; thenoyl; adamantane formoxyl; and mandeloyl;and X₂ is O or NH; or Ar₂ is

wherein each of R₃ and R₄ independently is any one of the followingsubstituent groups: H; alkyl; substituted alkyl which contains thesubstituent groups including halogen, alkoxyl or hydroxyl; C₂-C₆alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl; alkanoyl; substitutedalkanoyl which contains substituent groups including halogen, alkoxyl,or hydroxyl; C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl; benzoyl;tert-butoxycarbonyl; substituted benzoyl which contains optional one,two or three substituent groups including alkoxyl and alkylamino;benzyloyl; thenoyl; adamantane formoxyl; and mandeloyl; and X, is O orNH; X₂ is O or NH. When Ar₁ is

wherein each of R₅ and R₆ independently is any one of the followingsubstituent groups: H; alkyl; substituted alkyl which containssubstituent groups including halogen, alkoxyl, or hydroxyl; C₂-C₆alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl; alkanoyl; substitutedalkanoyl which contains substituent groups including halogen, alkoxyl orhydroxyl; C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl; benzoyl; substituted benzoylwhich contains optional one, two or three substituent groups includingalkoxyl and alkylamino; tert-butoxycarbonyl; benzyloyl; thenoyl;adamantane formoxyl; and mandeloyl; when X1 is O or NH; and X₂ is O orNH, Ar₂ is

wherein R₂ is any one of the following substituent groups: H; alkyl;substituted alkyl which contains substituent groups including halogen,alkoxyl or hydroxyl; C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl;alkanoyl; substituted alkanoyl which contains substituent groupsincluding halogen, alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆cycloalkanoyl, benzoyl, substituted benzoyl which contains optional one,two, or three substituent groups including alkoxyl and alkylamino;tert-butoxycarbonyl; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and X₂ is O or NH; or Ar₂ is

wherein each of R₃ and R₄ independently is any one of the followingsubstituent groups respectively: H; alkyl; substituted alkyl whichcontains substituent groups including halogen, alkoxyl or hydroxyl;C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl; alkanoyl; substitutedalkanoyl which contains substituent groups including halogen, alkoxyl,or hydroxyl; C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl, benzoyl, substitutedbenzoyl which contains optional one, two, or three substituent groupsincluding alkoxyl and alkylamino; tert-butoxycarbonyl; benzyloyl;thenoyl; adamantane formoxyl; and mandeloyl; and X, is O or NH; X₂ is Oor NH.

The present invention is performed by the following steps.

According to the chemical equation:

wherein each of Ar₁ and Ar₂ independently is phenyl or substitutedphenyl, and the substituent groups of the said substituted phenyl isone, two or three groups optionally selected from the following group:nitro; carboxyl; aldehyde; tert-butoxycarbonyl and thenoyl substitutedwith oxygen or amino; X is O, S or NH; and Y is O or S.

Or, according to the following chemical equation:

wherein R₁, R₂ and R₃ are optional any one of the following substitutentgroup: H; alkyl; substituted alkyl which contains substituent groupsincluding halogen, alkoxyl, or hydroxyl; C₂-C₆ alkenyl; C₃-C₆cycloalkyl; phenyl; benzyl; alkanoyl; substituted alkanoyl whichcontains substituent groups including halogen, alkoxyl, or hydroxyl;C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl; benzoyl; tert-butoxycarbonyl;substituted benzoyl which contains arbitrary one, two, or threesubstituent groups including alkoxyl and alkylamino; benzyloyl; thenoyl;adamantane formoxyl; X is O, S, or NH; Y is O or S; each of X₁, X₂ andX₃ independently is O or NH; and X₄ is Cl or OH.

The compound III is produced by condensing the compounds I with II. And,the condensation is performed in the following solvent: dichloromethane,acetic anhydride, tetrahydrofuran, dimethylfuran, dichloroethane,toluene, benzene, water, dioxane or the mixture of the above solvents.If necessary, some activators may be added into the reaction, such aspyridine, N-methylmorpholine, isobutyl chloroformate, triethylamine,diethylpropylethyl amine, or DMAP etc. According to reaction conditionsof the compounds, the reaction temperature generally is −78° C. to theroom temperature (for example, for the compound Wng462 etc.), or is 50□to 230□ by heating (for example, for the compound Wng520 etc). Thereaction time is determined according to the specific reactants.Generally, the reaction progress is determined by tracing with TLC.After the completion of the reaction, the general post processingmethods include filtrating with a pump, concentrating the reactionsolution to remove the solvent, extracting and isolating with columnchromatography etc. The final product III is verified with NMRdetection.

The process for synthesizing the structural unit of the substitutedfive-membered heterocyclic ring of the present invention refers toOrganic Syntheses, CV 2, 55.

The present invention designs and synthesizes the novel glucagon-likepeptide-1 receptor (GLP-1R) agonists. The GLP-1R agonists of the presentinvention have the good capability of binding the GLP-1R, promote thesynthesis of cAMP, may be used to prepare the medicaments for treatingthe glycometabolism disturbance-related diseases such as type IIdiabetes, insensitivity to insulin and obesity etc. And, they canovercome the defect that the polypeptides regulator medicaments isinconvenient for oral amnistration and readily to degrade in the priorart. The compounds of the present invention have the relative simplestructure and are readily to be prepared.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIG. 1 shows the detection result of the expression of the reportgene for the compounds of the present invention, which is used toevaluate the activating activity of the said compounds on GLP-1R. In theFIG. 1, the relative activity of the luciferase induced by 30 nM ofpositive standard GLP-1 is regarded as 100%.

The FIG. 2 shows the affection of the compound 2f on the concentrationof cAMP in 293/GLP-1R cells.

EMBODIMENTS OF THE PRESENT INVENTION

The present invention will be further explained with reference to thefollowing specific examples, but they don't limit the present inventionin any way.

The preparation process for preparing the compounds in the followingpreparation examples 1 to 3 mainly includes three reaction operationprocedures as follow.

Procedure 1:

The compounds I and II, sodium acetate, and acetic anhydride are mixed,heated to melt (150□ to 230° C.), and maintained in the molten state for1 hour. Subsequently, ethanol is added into the reaction mixture andcooled. The product is separated out by crystallization following byfiltration. The residue liquid is concentrated to remove the solventscompletely, and the product is isolated with column chromatography.

Procedure 2:

The compound I is dissolved in dichloromethane, and cooled in thecryohydrate bath at −20° C. following by adding trifluoroacetic acid andraising the temperature to the room temperature. And, the reaction istraced with TLC until the compound I is reacted completely. Afterconcentrating the reaction system and removing trifluoroacetic acidcompletely, the substrate is dissolved in dichloromethane, and cooled inthe cryohydrate bath at −20□. Then, pyridine and acyl chloride are addedorderly, the temperature is raised to the room temperature, and thereaction is traced with TLC. The reaction solution is concentrated, andthe product is isolated with column chromatography.

Procedure 3:

The compound I is dissolved in dichloromethane, and cooled in thecryohydrate bath at −20° C. following by adding trifluoroacetic acid andraising the temperature to the room temperature. And, the reaction istraced with TLC until the compound I is reacted completely, following byconcentrating the reaction system and removing trifluoroacetic acidcompletely. Then, the compound II is dissolved in tetrahydrofuran (THF),and cooled in the cryohydrate bath at −20□. Then, N-methylmorpholine(NMM) and ClCOO^(i)Bu are added orderly. The reaction product of thecompound I with trifluoroacetic acid is dissolved in tetrahydrofuran andthen transferred into the above mixture with the syringe so as to reactat the room temperature. The reaction is traced with TLC. After thereaction is completed, the reaction solution is concentrated, and theproduct is isolated with column chromatography.

For the products, the compounds wang520, wang337, wang405, wang450,wang520-1 and wang462-1 are prepared by the reaction procedure 1, thecompounds wang420, wang462, wang524, wang516, wang488, wang 568,wang502, wang530, wang504, wang866, 2f, wang582, wang538, and wang496are prepared by the procedure 2 with the compound wang520, and thecompounds wang516-1, and wang591 are prepared by the procedure 3 withthe compound wang520.

In the following preparation examples, NMR is measured with Mercury-Vx300M manufactured by Varian cooperation. NMR criteria are δH/C 7.26/7.77ppm(CDCl₃); δH/C 2.50/39.51 ppm (DSMO-d6); andδH/C 3.31/49.15 ppm(Methyl-d3 Alcohol-d). The agents are provided by Shanghai ChemistryAgents Cooperation. And, the products are purified mainly by the columnchromatography. The silica gel for separation is 200-300 mesh, and themodel of the silica gel for the column chromatography is thick andhollow (ZLX-II), and is produced by the branch factory of QingdaoHaiyang Chemical plant.

EXAMPLE 1

At the room temperature, the compound II (466 mg, 1.78 mmol), thecompound I (576 mg, 1.96 mmol), sodium acetate (146 mg, 1.78 mmol) and 2mL of acetic anhydride are mixed, following by heating to 170° C. untilthe system melts, and maintaining in the molten state for 1 hour. Then,2 mL of ethanol is added into the resultant mixture, and then cooled tothe room temperature. So the yellow solids are separated out andfiltered. The residue liquid is concentrated, and the solvent is removedcompletely, to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (5:1 v/v) to obtain 556 mg of product, the compound wang520(yield: 60%).

¹HNMR (300 MHz; CDCl₃) δ 1.54 (s,9H),3.95 (s,3H), 6.79 (br, 1H), 7.16(s, 1H), 7.20 (dd, J=4.8 Hz, 3.9 Hz, 1H), 7.25 (d, J=9.9 Hz, 1H), 7.53(d, J=9.0 Hz, 2H), 7.63 (dd, J=8.4 Hz, 2.1 Hz, 1H), 7.69 (dd, J=4.8 Hz,1.2 Hz, 1H), 8.02 (dd, J=3.9 Hz, 1.2 Hz, 1H), 8.06 (d, J=8.7 Hz, 2H),8.17 (d, J=1.5 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 28.17, 55.79, 81.23,115.28, 117.92, 119.11, 123.09, 125.74, 128.02, 129.29, 129.41, 132.18,132.75, 133.29, 133.71, 134.99, 141.57, 143.46, 151.37, 152.08, 159.93,163.13, 167.46.

At the room temperature, the compound II (466 mg, 1.78 mmol), thecompound I (576 mg, 1.96 mmol), sodium acetate (146 mg, 1.78 mmol) and 2mL of acetic anhydride are mixed, following by heating to 200° C. untilthe system melts, and maintaining in the molten state for 1 hour. Then,2 mL of ethanol is added into the resultant mixture, and then cooled tothe room temperature. The liquid is concentrated, and the solvent isremoved completely, to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (1:1 v/v) to obtain 158 mg of the compound wang462-1.

¹H NMR (300 MHz, CDCl₃, wang520-1) δ 1.50 (s, 9H), 3.88 (s, 3H), 7.27(s, 1H), 7.33-7.37 (2H), 7.69 (d, J=8.7 Hz, 2H), 8.01 (d, J=8.7 Hz, 2H),8.07 (d, J=3.9 Hz, 1H), 8.13 (d, J=4.8 Hz, 1H), 8.22-8.26 (2H), 9.93 (s,1H). ¹H NMR (300 MHz, CDCl₃, wang462-1) δ 2.22 (s, 3H), 3.91 (s, 3H),7.07 (d, J=8.7 Hz, 1H), 7.14 (s, 1H), 7.21 (m, 1H), 7.42 (m, 1H), 7.66(d, J=8.1 Hz, 2H), 7.71 (d, J=4.8 Hz, 1H), 7.99 (d, J=8.7 Hz, 1H), 8.05(m, 1H), 8.10 (d, J=8.4 Hz, 2H, 8.19 (m, 1H).

At the room temperature, the compound II (1.46 g, 9.6 mmol), thecompound I (1.9 g, 10.7 mmol), sodium acetate (0.8 g, 9.8 mmol) and 2.8mL of acetic anhydride are mixed, following by heating to 170° C. untilthe system melts, and maintaining in the molten state for 1 hour. Then,5 mL of ethanol is added into the resultant mixture, and then cooled tothe room temperature. So the yellow solids are separated out andfiltered to obtain 2.0 g of product, the compound wang337 (yield: 62%).

¹HNMR (300MHz, CDCl₃) δ 2.35 (s, 3H), 3.97 (s, 3H), 7.13 (d, J=8.4 H1H),7.20 (s, 1H), 7.50-7.56 (2H), 7.59-7.65 (2H), 8.12-8.15 (3H).

At the room temperature, the compound II (262 mg, 1.0 mmol), thecompound I (200 mg, 1.1 mmol), sodium acetate (82 mg, 1.0 mmol) and 1 mLof acetic anhydride are mixed, following by heating to 170° C. until thesystem melts, and maintaining in the molten state for 1 hour. Then, 5 mLof ethanol is added into the resultant mixture, and then cooled to theroom temperature. So the yellow solids are separated out and filtered.The residue liquid is concentrated, and the solvent is removedcompletely, to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (6:1 v/v) to obtain 235 mg of product, the compound wang405(yield: 58%).

¹HNMR (300MHz, CDCl₃) δ 3.97 (s,3H), 7.20 (dd, J4.8 Hz, 3.9 Hz, 1H),7.24 (s 1H), 7.26 (d, J=7.8 Hz, 1H), 7.51-7.57 (2H), 7.60-7.70 (3H),8.02 (dd, J=3.6 Hz, 0.9 Hz, 1H), 8.14-8.19 (3H).

At the room temperature, the compound II (262 mg, 1.0 mmol), thecompound I (250 mg, 1.1 mmol), sodium acetate (82 mg, 1.0 mmol) and 4 mLof acetic anhydride are mixed, following by heating, and maintaining thesystem at 210 to 230° C. for 1 hour. Then, 5 mL of ethanol is added intothe resultant mixture, and then cooled to the room temperature. So theyellow solids are separated out and filtered to obtain 100 mg ofproduct, the compound wang450 (yield: 22%).

¹HNMR (300 MHz, CDCl₃) δ 3.97 (s,3H), 7.21 (dd, J=4.8 Hz, 3.9 Hz, 1H),7.30 (d, J=3.9 Hz, 1H), 7.37 (s, 1H), 7.70 (d, J=5.1 Hz, 1H), 7.73 (dd,J=9.9 Hz, 1.5 Hz, 1H), 8.02 (d, J=3.9 Hz, 1H), 8.09 (d, J=1.8 Hz, 1H),8.33 (d, J=9.0 Hz, 2H), 8.40 (d, J=9.3 Hz, 2H).

EXAMPLE 2

The compound I (50 mg, 0.1mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20° C.following by adding 1 mL of trifluoroacetic acid and gradually raisingthe temperature to the room temperature. And, the reaction is tracedwith TLC until the compound I is reacted completely. After concentratingthe reaction system and removing trifluoroacetic acid completely, thenthe reaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, and gradually raising the temperature to the roomtemperature. The reaction is traced with TLC. The reaction solution isconcentrated, and the solvents are removed to obtain the crude product.The crude product is chromatographed over silica gel column withpetroleum ether/ethyl acetate (2:1 v/v) to obtain 38 mg of product, thecompound wang420 (yield: 90%).

¹HNMR (300 MHz, CDCl₃) δ 3.94 (s, 3H), 7.20-7.24 (m, 2H), 7.27 (d, J=1.8Hz, 1H), 7.66 (dd, J=8.1 Hz, 1.5 Hz, 1H), 7.71 (dd, J=4.8 Hz, 0.9 Hz,1H), 7.76 (d, J=9.0 Hz, 2H), 8.03 (dd, J=3.6 Hz, 0.9 Hz, 1H), 8.07 (d,J=1.5 Hz, 1H), 8.14(d, J=8.7 Hz, 2H), 8.20 (br, 2H).

The compound I (50 mg, 0.1 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20° C.following by adding 1 mL of trifluoroacetic acid and gradually raisingthe temperature to the room temperature. And, the reaction is tracedwith TLC until the compound I is reacted completely. After concentratingthe reaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (27 μL, 0.39 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (1.5:1 v/v) to obtain 26 mg of product, the compound wang462(yield: 56%).

¹H NMR (300 MHz CDCl₃) δ 2.19 (s, 3H), 3.88 (s, 3H), 7.12 (s, 1H),7.20-7.24 (m, 2H), 7.55 (d, J=1.5 Hz, 1H), 7.60 (d, J=9.0 Hz,2H), 7.71(dd, J=4.8 Hz, 0.9 Hz, 1H), 7.77 (br, 1H), 7.97 (d, J=8.7 Hz, 2H), 8.03(dd, J=3.9 Hz, 0.9 Hz, 1H), 8.07 (d, J=1.5 Hz, 1H), ¹³C NMR (75 MHzCDCl₃) δ 24.66, 55.84, 155.64, 119.55, 120.54, 123.35, 126.15, 128.43,129.59, 129.87, 13237, 133.12, 133.52, 134.26,135.41, 141.85, 143.13,151.63, 160.63, 163.28, 167.60, 168.99.

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20° C.following by adding 1 mL of trifluoroacetic acid-and gradually raisingthe temperature to the room temperature. And, the reaction is tracedwith TLC until the compound I is reacted completely. After concentratingthe reaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (23 μL, 0.2 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (5:1 v/v) to obtain 15 mg of product, the compound wang524(yield: 36%).

¹HNMR (300 MHz DMSO-d₄) δ 3.90 (s, 3H), 7.22 (d, J=5.4 Hz, 1H), 7.33 (d,J=8.4 H 2H), 7.39-7.44 (1H), 7.50-7.58 (2H), 7.83 (d, J=8.4 Hz), 7.98(d, J=8.7 Hz, 2H), 8.04-8.22 (7H), 10.74 (s, 1H).

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20° C.following by adding 1 mL of trifluoroacetic acid and gradually raisingthe temperature to the room temperature. And, the reaction is tracedwith TLC until the compound I is reacted completely. After concentratingthe reaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (25 μL, 0.2 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (4:1 v/v) to obtain 25 mg of product, the compound wang516(yield: 62.5%).

¹H NMR (300 MHz DMSO-d₆) δ 1.57 (m, 2H), 1.63-1.77 (m, 4), 1.80-1.89 (m,2H), 2.84 (m, 1H), 3.89 (s, 3H), 7.31 (m, 2H), 7.40 (d, J=8.4 Hz, 1H),7.86 (d, J=9.0 Hz, 2H), 7.94 (dd, J=8.4 Hz, 1.8 Hz, 1H), 8.03 (dd, J=3.9Hz, 1.2 Hz, 1H), 8.07 (d, J=9.0 Hz, 2H), 8.10 (dd, J=4.8 Hz, 1.2 Hz,1H), 8.18 (d, J=1.8 Hz, 1H), 10.35 (s, 1H); ¹³C NMR (75 MHz, DMSO₆) δ25.62, 30.00, 55.97, 115.74, 118.71, 119.04, 123.52, 125.27, 128.51,128.77, 129.24, 131.19, 132.78. 133.34, 135.43, 135.50, 140.86, 144.42,151.04, 159.24, 162.91, 166.93, 175.11.

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20° C.following by adding 1 mL of trifluoroacetic acid and gradually raisingthe temperature to the room temperature. And, the reaction is tracedwith TLC until the compound I is reacted completely. After concentratingthe reaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (23 μL, 0.2 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (4:1 v/v) to obtain 25 mg of product, the compound wang488(yield: 64%).

¹H NMR (300 MHz DMSO-d₆) δ 0.80 (m, 2H), 0.85 (m, 2H), 1.84 (m, 1H),3.88 (s,3H), 7.28 (s, 1H), 7.32 (dd, J=5.1 Hz 3.9 Hz, 1H), 739 (d, J=8.1Hz, 1H), 7.85 (d, J=8.7 Hz, 2H), 7.92 (dd, J=8.4 Hz, 1.5 Hz, 1H), 8.04(m, 1H), 8.05 (d, J=8.7 Hz, 2H), 8.11 (dd, J=4.8 Hz, 1.2Hz, 1H, 8.18 (d,J=1.8 Hz, 1H), 10.68 (s, 1H); ³C NMR (75 MHz, DMSO-d₆) δ 7.78, 14.83,55.97, 115.71, 118.73, 118.93, 123.53, 12532, 128.54, 128.81, 129.32,131.22, 132.80, 133.36, 135.46, 135.53, 140.88, 144.24, 151.05, 159.29,162.91, 166.96, 172.44.

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20□ followingby adding 1 mL of trifluoroacetic acid and gradually raising thetemperature to the room temperature. And, the reaction is traced withTLC until the compound I is reacted completely. After concentrating thereaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (23 μL, 0.2 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (4:1 v/v) to obtain 26 mg of product, the compound wang568(yield: 57%).

¹H NMR (300 MHz, CDCl₃) δ 3.95 (s, 3H), 4.13 (s, 2H), 4.68 (s, 2H), 7.18(s, 1H), 7.19-7.26 (m, 2H), 7.39-7.50 (m, 5H), 7.63 (dd, J=6.9 Hz, 0.9Hz, 1H), 7.69 (dd, J=4.8 Hz, 0.9 Hz, 1H), 7.74 (d, J=9.0 Hz, 2H), 8.01(dd, J=3.6 Hz, 1.2 Hz, 1H), 8.10 (d, J=8.7 Hz, 2H), 8.16 (d, J=1.5 Hz,1H), 8.56 (s, 1H).

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20□ followingby adding 1 mL of trifluoroacetic acid and gradually raising thetemperature to the room temperature. And, the reaction is traced withTLC until the compound I is reacted completely. After concentrating thereaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (23 μL, 0.2 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (4:1 v/v) to obtain 22 mg of product, the compound wang502(yield: 56%).

-   -   ¹H NMR (300 MHz, DMSO-d₆) δ 1.81-1.94 (m, 2H), 2.12-2.28 (m,        4H), 3.29 (m, 1H), 3.89 (s, 3H), 7.31 (s, 1H), 7.33 (m, 1H),        7.40 (d, J=7.5 Hz, 1H), 7.87 (d, J=8.1 Hz, 2H), 7.94 (d, J=8.1        Hz, 2H), 8.04-8.08 (2H), 8.12 (d, J=5.1 Hz, 1H), 8.19 (s, 1H),        10.20 (s, 1H).

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20□ followingby adding 1 mL of trifluoroacetic acid and gradually raising thetemperature to the room temperature. And, the reaction is traced withTLC until the compound I is reacted completely. After concentrating thereaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (23 μL, 0.2 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (4:1 v/v) to obtain 24 mg of product, the compound wang530(yield: 57%).

¹H NMR (300 MHz, DMSO-₄) δ 120-1.48 (6H), 1.65-1.81 (4H), 2.39 (m, 1H),3.89 (s, 3H), 7.32 (s, 1H), 7.34 (m, 1H), 7.41 (d, J=8.4 MHz, 1H), 7.87(d, J=8.1 Hz, 2H), 7.95 (d, J=8.1 Hz. 1H), 8.04 (m, 1H), 8.08 (d, J=8.7Hz, 2H), 8.12(d, J=4.8 HZ, 1H), 8.20(m, 1H), 10.31 (s, 1H).

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20□ followingby adding 1 mL of trifluoroacetic acid and gradually raising thetemperature to the room temperature. And, the reaction is traced withTLC until the compound I is reacted completely. After concentrating thereaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (23μL, 0.2 mmol) and graduallyraising the temperature to the room temperature. The reaction is tracedwith TLC. The reaction solution is concentrated, and the solvents areremoved to obtain the crude product. The crude product ischromatographed over silica gel column with petroleum ether/ethylacetate (6:1 v/v) to obtain 4 mg of product, the compound wang504(yield: 10%).

¹H NMR (300 MHz, CDCl₃) δ 1.34 (s,9 H), 3.94 (s,3H), 7.15 (s, 1H), 720(dd, J=4.8 Hz, 3.6 Hz, 1H), 7.23 (s, 1H),7.58 (br, 1H), 7.64-7.69 (2H),7.72 (d, J=8.7 Hz, 2H), 8.02 (dd, J=3.6 Hz, 1.5 Hz, 1H), 8.08 (d, J=9.0Hz,2H),8.11(d, J=1.8 Hz, 1H).

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20□ followingby adding 1 mL of trifluoroacetic acid and gradually raising thetemperature to the room temperature. And, the reaction is traced withTLC until the compound I is reacted completely. After concentrating thereaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (27μL, 0.2 mmol) and graduallyraising the temperature to the room temperature. The reaction is tracedwith TLC. The reaction solution is concentrated, and the solvents areremoved to obtain the crude product. The crude product ischromatographed over silica gel column with CH₂Cl₂ to obtain 40 mg ofproduct, the compound wang554 (yield: 89%).

¹H NMR(300 MHz, CDCl₃) δ 3.83 (s, 3H), 6.28 (s, 1H), 7.05 (s, 1H), 7.16(d, J=8.1 Hz, 1H), 7.20 (dd, J=5.1 Hz, 3.6 Hz, 1H), 7.39-7.41 (2H),7.50-7.55 (3), 7.60 (d, J=9.0 Hz, 2H), 7.71 (dd, J=5.1 Hz, 1.2 Hz, 1H),7.92 (d, J=8.4 Hz, 2H), 7.99 (d, J=1.2 Hz), 8.03 (dd, J=3.6 Hz, 0.9 Hz,2H), 8.24 (s, 1H), 8.42 (s, 1H).

The compound I (52 mg, 0.1 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20□ followingby adding 1 mL of trifluoroacetic acid and gradually raising thetemperature to the room temperature. And, the reaction is traced withTLC until the compound I is reacted completely. After concentrating thereaction system and removing trifluoroacetic acid completely, thereaction intermediate is dissolved in 2 mL of dichloromethane, andcooled in the cryohydrate bath at −20□, following by adding 40 μL (0.6mmol) of pyridine, adding the compound II (10 mg, 0.03 mmol) andgradually raising the temperature to the room temperature. The reactionis traced with TLC. The reaction solution is concentrated, and thesolvents are removed to obtain the crude product. The crude product ischromatographed over silica gel column with CH₂Cl₂ to obtain 19 mg ofproduct, the compound wang866 (yield: 44%).

¹H NMR (300 MHz, DMSO-d₄) δ 3.89 (s, 6H), 7.33 (dd, J=4.8 Hz, 3.9 Hz,2H), 7.36 (s 2H), 7.41 (d, J=8.4 Hz, 2H), 7.93 -7.96 (2H), 7.96 (d,J=8.7 Hz, 4H), 8.04 (dd, J=3.3 Hz, 0.9 Hz, 2H), 8.12 (dd, J=4.8 Hz, 0.9Hz, 2H), 8.17 (d, J=8.7 Hz, 4H), 8.20 (d, J=1.8 Hz, 2H), 11.66 (s, 2H).

Accord to the same process, the compound 2f is prepared by using thereaction product of 1 eq of compound

with trifluoroacetic acid and 1.5 eq of acetyl chloride (yield: 56%). ¹HNMR (300 MHz, CDCl₃) δ 1.41 (t, J=6.9 Hz, 3H), 2.24 (s, 3H), 4.18 (q,J=6.9 Hz, 2H), 7.11 (s, 1H), 7.19 (m, 1H), 7.45 (m, 2H), 7.62-7.70 (4H),8.02 (m, 1H), 8.08 (d, J=9.0 Hz, 2H).

According the same process, the compound wang582 is prepared by usingthe reaction product of 1 eq of the compound wang520 withtrifluoroacetic acid and 1.5 eq of diamantane formyl chloride (yield:38%).

¹H NMR (300 MHz, CDCl₃) δ 1.76 (6H), 1.99 (6H), 2.12 (3H), 3.95 (s, 3H),7.14-7.23 (2H), 7.54 (s, 1H), 7.61-7.70 (2H), 7.73 (d, J=9.0 Hz, 2H),8.02 (dd, J=3.9 Hz, 1H), 8.09 (d, J=9.0 Hz, 2H), 8.12 (d, J=1.8 Hz, 1H).

According the same process, the compound wang538 is prepared by usingthe reaction product of 1 eq of the compound wang520 withtrifluoroacetic acid and 1.5 eq of benzyl acetyl chloride (yield: 58%).

¹H NMR (300 MHz, CDCl₃) δ 3.78 (s, 2H), 3.92 (s, 3H), 7.16 (s, 1H),7.19- 7.24 (2H), 7.34-7.74 (6H), 7.59 (d, J=8.7 Hz, 214, 7.62 (m, 1H),7.70 (d, J=4.8 Hz, 1H), 8.02 (d, J=8.7 Hz, 2H), 8.13 (m, 1H).

According the same process, the compound wang496 is prepared by usingthe reaction product of 1 eq of the compound wang520 withtrifluoroacetic acid and 1.5eq of chloro acetyl chloride (yield: 70%).

¹H NMR (300 MHz, DMSO-d₆) δ 3.89 (s, 3H), 4.36 (s, 2H), 7.34 (s,1H),7.41 (d, J=8.1 Hz, 1H), 7.88 (d, J=9.0 Hz, 2H), 7.93-7.98 (2H), 8.05(m, 1H), 8.12 (d, J=7.5 Hz, 2H), 8.22 (m, 1H), 10.95 (s, 1H.

EXAMPLE 3

The compound I (40 mg, 0.08 mmol) is dissolved into 2 mL ofdichloromethane, and is cooled in the cryohydrate bath at −20□ followingby adding 1 mL of trifluoroacetic acid and gradually raising thetemperature to the room temperature. And, the reaction is traced withTLC until the compound I is reacted completely. After concentrating thereaction system and removing trifluoroacetic acid completely, thecompound II (19 μL, 0.16 mmol)is dissolved in 2 mL of tetrahydrofuran,and cooled in the cryohydrate bath at −20□ with stirring for 10 min atthis temperature. Then, N-methylmorpholine (NMM) (53 μL, 0.48 mmol) andClCOO^(i)Bu (21 μL, 0.16 mmol) are added orderly with stirring for 0.5hour at −20□. The reaction product of the compound I withtrifluoroacetic acid is dissolved in 1 mL of tetrahydrofuran and thentransferred into the above mixture with the syringe so as to react atthe room temperature for about 15 hours. The reaction solution isconcentrated and the solvents are removed completely to obtain the crudeproduct. The crude product is chromatographed over silica gel columnwith petroleum ether/ethyl acetate (5:1 v/v) to obtain 12 mg of product,the compound wang516-1(yield: 30%).

¹HNMR(300 MHz, CDCl₃) δ 1.74 (s, 3H), 1.87 (s, 3H),3.18 (d,J=7.8 Hz,2H), 3.95 (s, 3, H), 5.42 (m, 1H), 7.19 (s, 1H), 7.20-7.27 (2H), 7.63(2H), 7.65 (d, J=1.8 Hz, 1H), 7.70 (d, J=8.4 Hz, 2H), 8.02 (dd, J=3.6Hz, 0.9 Hz, 1H), 8.09 (d, J=9.0 Hz, 2H), 8.16 (d, J=2.1 Hz, 1H).

According the same process, the compound wang591 is prepared by usingthe reaction product of 1 eq of the compound wang520 withtrifluoroacetic acid and 2.0 eq of the compound Boc-Ala-OH (yield: 18%).

EXAMPLE 14

The compound wang568 (11 mg, 0.02 mmol) is dissolved into 2 mL ofdichloromethane, and cooled at −78□ for 10 minutes, following by adding0.2 mL of BCl₃/n-hexane solution (1M) to continue reacting for 30minutes at −78□. Then, the temperature is raised to −18□ to react for 4hours. 2 mL of ether is added to quench the reaction with stirring for30 minutes at the room temperature, following by adding 5 mL of water.The water phase and the organic phase are separated. The water phase isextracted with dichloromethane, and the organic phase is combined, driedwith anhydrous MgSO₄, and concentrated. The crude product is isolatedover column chromatography with petroleum ether/ethyl acetate (1/2, v/v)to obtain the compound wang477 (1.5 mg, yield: 17%).

¹H NMR (300 MHz, CDCl₃) δ 1.86 br, 1H, 3.95 (s, 3H), 4.26 (s, 2H), 7.18(s, 1H), 7.20 (dd, J=8.7 Hz, 4.8 Hz, 1H), 7.23 (d, J=3.3 Hz, 1H), 7.63(d, J=8.1 Hz, 1H), 7.71 (dd, J=5.1 Hz, 1.5 Hz, 1H), 7.75 (d, J=8.7Hz,2H), 8.02 (d, J=3.6 Hz, 1H), 8.08 (d, J=8.7 Hz, 2H), 8.14 (m, 1H),8.57 b r, 1H).

The compound wang591 (3 mg) is dissolved into 1.5 mL of dichloromethane,and cooled in ice bath for 5 minutes, following by adding 0.15 mL oftrifluoroacetic acid. Then, the temperature is gradually raised to theroom temperature, and the reaction is traced with TLC. After the rawmaterial disappears, the solvent and trifluoroacetic acid are removed bypumping to obtain 2 mg of the product, the compound wang 605 (yield:65%).

¹H NMR (300 MHz, Methyl-d₃ Alcohol-d) δ 1.63 (d, J=7.2 Hz, 3H),3.95(s,3H), 4.09 (m, 1H), 7.265 (s,1H), 7.267 (d, J=8.7 Hz, 1H), 7.29 (d,J=8.1 Hz, 1H),7.81 (dd, J=8.7 Hz, 2.1 Hz, 1H), 7.87 (d, J=9.0 Hz, 2H),7.91 (dd, J=5.1 Hz, 1.2 Hz, 1H), 8.01 (dd, J=3.6 Hz, 0.9 Hz, 1H), 8.16(d, J=9.3 Hz, 2H), 8.25 (d, J=2.1 Hz, 1H).

EXAMPLE 4 Experiments Testing on Biological Activity

1. Testing the Expression of the Report Gene

Upon that GLP-1R binds to GLP-1 or agonists, its Gasubunit is activatedto stimulate the adenylate cyclase, which makes the increase in theconcentration of intracellular cAMP. Since the promoter region of theproinsulin gene has the cAMP response element, upon binding of cAMP tothis response element, the transcription of the proinsulin gene isactivated, so as to increase the sensitivity of -- islet cells toglucose, and improve the expression and secretion of insulin (Diabetes,2000, vol.49:1156-1164). The screening model employs the human embryonalnephric cell strain which is stably transfected with the expressionvector of GLP-1R gene and the expression vector of luciferase reportgene under the regulation of cAMP response element, to detect itsresponse to the candidate compound (Cell Biology, 1992, Vol.89:8641-8645; Proc. Natl. Acad. Sci. U.S.A. 1987, Vol.84: 3434-3438). Whenscreening the candidate compounds, the compound which may induce theluciferase report gene to express have the activity of activating GLP-1.

1.1 Experimental Material and Instruments

Cell strain: HEK 293/GLP1R+Luc strain which stably express GLP-1R andluciferase (National New Medicaments Screening Center)

Fetal calf serum (GIBCO/BRL Cooperation)

Steady-glo™ luciferase analysis system (Promega Cooperation)

Standard GLP-1 (Sigma Cooperation)

G418 (Invitrogen Cooperation)

Forma carbon dioxide incubator (Forma Cooperation)

Victor 2 counting machine (Wallac Cooperation)

Candidate compound: the compounds wang524, wang520, wang462, 2f,wang516, wang516-2, wang502 and wang504;

1.2 Experimental Process

HEK 293/GLP1R+Luc cell in 20000 cells/100 μl/well are inoculated into96-well plate, are cultured at 37□ overnight with DMEM culture mediumcontaining 10% of fetal calf serum and 500 μg/mL of G418. The candidatecompounds wang516-2, wang502, and wang504 are respectively diluted to 2mM, 1 mM, 0.3 mM, 0.1 mM, 0.03 mM, 0.1 mM, and 0.003 mM, and the othercandidate compounds are diluted gradually from 30 mM for 8 times by aratio of 1:3 to get a concentration gradient (i.e., 30 mM, 10 mM, 3 mM,1 mM, 0.3 mM, 0.1 mM, 0.03 mM, and 0.01 mM), following by being addedinto the above 96-well plate at 1 μl/well. Then, the cells are culturedat 37□ in 5% of CO₂for 6 hours. The activity of luciferase is detectedaccording to the specification of Steady-glo™ luciferase analysis systemkit, and counting is performed with Victor 2 counting machine. Thepositive control adopts 30 nM of standard GLP-1.

1.3 Experimental Result

The experimental result of the report gene for the candidate compoundsis as shown in the FIG. 1 and the table 1.

The FIG. 1 shows that the compound wang 520 in a final concentration of30 μM have the best relative activity (94%) which is improved greatlycompared with the activity of the compound 2f. In addition, thecompounds as shown in the table 1 have the dose dependency on theactivity of GLP-1R, wherein the median effective dose (EC₅₀) of thecompounds wang 520, wang 516. wang 554, wang 488 wang516-2, wang502 andwang 504 is less than 10 μM. Such result provides the direction fordetermining the superior structure of the interaction of the compoundswith GLP-1R. TABLE 1 The number of the compound EC₅₀/μM wang524 46.5wang520 4.6 wang462 11.6 wang516 6.85 2f 13.0 wang866 54.41 wang554 5.24wang488 6.73 wang516-2 6.06 wang502 3.31 wang504 4.87

2. Determination of the Concentration of Intracellular cAMP

Since the determination of the concentration of intracellular cAMPindirectly by detecting the expression of the report gene is an indirectprocess, the functional re-screen is directly performed with thecAMP-detecting kit in order to make sure that the compound can surelyincrease the concentration of intracellular cAMP.

2.1 Experimental Material and Instruments

cAMP-detecting kit (Applied Biosystems Cooperation)

Forma carbon dioxide incubator (Forma Cooperation)

Victor 2 according machine (Wallac Cooperation)

HEK 293/GLP1R+Luc strain which stably express GLP-1R and luciferase(National new medicaments screening center)

Candidate compound: the compound 2f

Standard cAMP (provided in the kit, Applied Biosystems Cooperation)

2.2 Experimental process

HEK 293 cells are inoculated into 96-well plate in 20000 cells/100μl/well, are cultured at 37□ overnight. The compound 2f is diluted to1.00E-03M, 1.00E-04M, 1.00E-05M, 1.00E-06M and 1.00E-07M with dimethylsulphoxide, following by being inoculated into the above 96-well platein 11/well and being cultured at 37□ with 5% of CO₂ for 1 hour. Theconcentration of intracellular cAMP is detected according to thespecification of cAMP-Screen Direct TM system kit.

2.3 Experimental Result

The determining result of the concentration of intracellular cAMP isshown in the FIG. 2. As shown in the FIG. 2, with increase of theconcentration of the compound 2f, the concentration of cAMP which isproduced under this stimulation shows an exponential increase. Thisindicates that the compound 2f has a certain effect on signaltransmission of GLP-1R as a GLP-1R agonist. When the concentration ofthe compound 2f increases to 30 μM and 100 μM, the concentration of cAMPshows the decreasing trend, which is caused by the cellulotoxic effectof the high concentration of the compound 2f.

3. The Test on the Ligand-Binding Activity

In order to determine the binding activity of the compound to theligand, the cells which highly express GLP-1R are prepared, GLP-1labelled with ¹²⁵I is used as the ligand, while adding into thecandidate compound. When the candidate compound binds the ¹²⁵I-labelledGLP-1 competitively, the isotope labels on the cell membrane reduce.Accordingly, the affinity of the candidate compound to the ligand can beevaluated (J Mol Endocrinol. 2000 Vol.25:321-35; J Biomol Screen, 2000Vol. 5:377-84).

3.1 Experimental Material and Instruments

HEK 293/GLP1R+Luc cell strain (National New Medicaments ScreeningCenter)

Labeled compound: ¹²⁵I-labelled GLP-1 (Amersham Biosciences Cooperation)

Wallac MicroBata work station (Perkin Elmer Cooperation)

TomTech cell collector (TomTec Cooperation)

The testing buffer solution:

20 mM of tris-HCl (pH 7.4) (Shanghai Shenggong biological engineeringtechnology LTD), 100 mM of NaCl (Shanghai Chemical agents Cooperation),15 mM of NaF (Shanghai Chemical agents Cooperation), 2 mM ofdeoxypyridoxine (Sigma Cooperation), 0.2 mM of phenylmethylsulfonylfluoride (Sigma Cooperation), aprotinin (Shanghai Chemical agentsCooperation) (1 μg/ml), and leupeptin (Shanghai Chemical agentsCooperation) (1 μg/ml).

The washing solution:

20 mM of tris-HCl (pH 7.4), 100 mM of NaCl, and 15 mM NaF

The scintillation liquid (Wallac Cooperation)

The candidate compound is diluted with dimethyl sulphoxide at theconcentration gradient of 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10,000nM, and 100,000 nM.

3.2 Experimental Process

10⁵ HEK 293/GLP1R+Luc cells in the logarithmic growth phase areincubated together with the ¹²⁵I-labelled GLP-1 positive peptide (thefinal concentration of 40 pM) in 200 μl of the testing buffer solutionat 25□ for 4 hours, while adding into the non-labeled positive peptideor the candidate compound. The cells are collected with the cellcollector, following by washing three times with the washing solution.The scintillation liquid is added into them, and each well is countedwith Microbata counter.

3.3 Experimental Result

The result of the receptor-binding experiment is shown in the table 3.As shown in the table 3, the compound 2f has the better affinity toGLP-1R, but the affinities of compounds wang520 and wang516 are littleweek, and the other compound substantially don't bind the receptor inthe testing concentration rage. TABLE 3 The number of the compoundEC₅₀/μM wang524 >100 μM wang450 >100 μM wang405 >100 μM wang327 >100 μMwang520 60-100 μM wang462 >100 μM wang866 >100 μM wang516 40-80 μMwang420 >100 μM 2f 31 μM

1. A glucagon-like peptide-1 receptor agonist having the followingstructural formula:

wherein, each of Ar₁ and Ar₂ independently is phenyl or substitutedphenyl, and the substituent groups of the said substituted phenyl isone, two or three groups optionally selected from the following group:alkyl; hydroxyl; substituted alkoxyl or alkylamino which contains thesubstituent groups including halogen, alkoxyl or hydroxyl; substitutedalkanoylxy or alkanoylamino which contains the substitutent groupsincluding halogen, alkoxyl or hydroxyl; C₂-C₆ alkenyl substituted withoxygen or amine, phenyl, benzyl, C₂-C₆ enoyl, C₃-C₆ cycloalkanoyl,benzoyl, substituted benzoyl which contains optional one, two or threesubstituent groups including alkoxyl and alkanoylamino, benzyloyl,thenoyl, tert-butoxycarbonyl, adamantane formoxyl, and mandeloyl;alkoxyl; alkylamino; cycloalkoxyl; cycloalkylamino; amino; amide;alkoxycarbonyl; cycloalkoxycarbonyl; alkanoylxy; al kanoylami no;cycloalkanoylxy; cycloalkanoylamino; carbamido; urylene; alkanoyl;nitro; carboxyl; and aldehyde group; X is O, S, or NH; and Y is O or S.2. The glucagon-like peptide-1 receptor agonist according to the claim1, being characterized in that when Ar₁ is

wherein R₁ is any one of the following substituent groups: H; alkyl;substituted alkyl which contains the substituent groups includinghalogen, alkoxyl or hydroxyl; C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl;benzyl; alkanoyl; substituted alkanoyl which contains the substituentgroups including halogen, alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆cycloalkanoyl; benzoyl; substituted benzoyl which contains optional one,two or three substituent groups including alkoxyl and alkylamino;tert-butoxycarbonyl; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and X₁ is O or NH, Ar₂ is

wherein R₂ is any one of the following substituent groups: H; alkyl;substituted alkyl which contains the substituent groups includinghalogen, alkoxyl or hydroxyl; C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl;benzyl; alkanoyl; substituted alkanoyl which contains the substituentgroups including halogen, alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆cycloalkanoyl; benzoyl; substituted benzoyl which contains optional one,two or three substituent groups including alkoxyl and alkylamino;tert-butoxycarbonyl; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and X₂ is O or NH; or Ar₂ is

wherein each of R₃ and R₄ independently is any one of the followingsubstituent groups: H; alkyl; substituted alkyl which contains thesubstituent groups including halogen, alkoxyl or hydroxyl; C₂-C₆alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl; alkanoyl; substitutedalkanoyl which contains the substituent groups including halogen,alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl; benzoyl;substituted benzoyl which contains optional one, two or threesubstituent groups including alkoxyl and alkylamino;tert-butoxycarbonyl; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and X₁ is O or NH; X₂ is O or NH.
 3. The glucagon-likepeptide-1 receptor agonist according to the claim 1, being characterizedin that, when Ar₁ is

wherein each of R₅ and R₆ independently is any one of the followingsubstituent groups: H; alkyl; substituted alkyl which contains thesubstituent groups including halogen, alkoxyl or hydroxyl; C₂-C₆alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl; alkanoyl; substitutedalkanoyl which contains the substituent groups including halogen,alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl; benzoyl;substituted benzoyl which contains optional one, two or threesubstituent groups including alkoxyl and alkylamino;tert-butoxycarbonyl; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and X₁ is O or NH; X₂ is O or NH, Ar₂ is

wherein R₂ is any one of the following substituent groups: H; alkyl;substituted alkyl which contains substituent groups including halogen,alkoxyl or hydroxyl; C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl;alkanoyl; substituted alkanoyl which contains substituent groupsincluding halogen, alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆cycloalkanoyl; benzoyl; substituted benzoyl which contains optional one,two or three substituent groups including alkoxyl and alkylamino;tert-butoxycarbonyl; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and X₂ is O or NH; or Ar₂ is

wherein each of R₃ and R₄ independently is any one of the followingsubstituent groups: H; alkyl; substituted alkyl which containssubstituent groups including halogen, alkoxyl or hydroxyl; C₂-C₆alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl; alkanoyl; substitutedalkanoyl which contains the substituent groups including halogen,alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl; benzoyl;substituted benzoyl which contains optional one, two or threesubstituent groups including alkoxyl and alkylamino;tert-butoxycarbonyl; benzyloyl; thenoyl; adamantane formoxyl; andmandeloyl; and Xi is O or NH; X₂ is O or NH.
 4. A process for preparingthe glucagon-like peptide-1 receptor agonist according to the claim 1,being characterized in that, the said agonist is prepared bycondensating the compound

and Ar₁CHO, wherein each of Ar₁ and Ar₂ independently is phenyl orsubstituted phenyl, wherein the substituent group of the saidsubstituted phenyl is one, two or three groups optionally selected fromthe following group: nitro; carboxyl; aldehyde; tert-butoxycarbonyl andthenoyl substituted with oxygen or amino; X is O, S or NH; and Y is O orS.
 5. A process for preparing the glucagon-like peptide-1 receptoragonist according to the claim 1, being characterized in that, thecompound

is prepared by condensating the reaction product of compound

and trifluoroacetic acid with the compound R₁COX₄, wherein R₁, R₂ and R₃are any one of the following substitutent groups: H; alkyl; substitutedalkyl which contains the substituent groups including halogen, alkoxylor hydroxyl; C₂-C₆ alkenyl; C₃-C₆ cycloalkyl; phenyl; benzyl; alkanoyl;substituted alkanoyl which contains the substituent groups includinghalogen, alkoxyl or hydroxyl; C₂-C₆ enoyl; C₃-C₆ cycloalkanoyl; benzoyl;tert-butoxycarbonyl; substituted benzoyl which contains optional one,two or three substituent groups including alkoxyl and alkylamino;benzyloyl; thenoyl; adamantane formoxyl; and mandeloyl; X is O, S, orNH; Y is O or S; each of X₁, X₂ and X₃ independently is O or NH; and X4is Cl or OH.
 6. The processes for preparing the glucagon-like peptide-1receptor agonist according the claims 4 or 5, being characterized inthat, the solvent used in condensation reaction is dichloromethane,acetic anhydride, tetrahydrofuran, dimethylfuran, dichloroethane,toluene, benzene, water, dioxane or any mixture thereof.
 7. Theprocesses for preparing the glucagon-like peptide-1 receptor agonistaccording the claims 4 or 5, being characterized in that, the reactiontemperature is from −78° C. to the room temperature, or the heatingtemperature is from 50□ to 230□.
 8. The processes for preparing theglucagon-like peptide-1 receptor agonist according the claims 4 or 5,being characterized in that, pyridine, triethylamine, diethylpropylethylamine, DMAP, N-methylmorpholine, or isobutyl chloroformate is used asactivator in condensation reaction.
 9. Use of the glucagon-likepeptide-1 receptor agonist according to claim 1 as medicaments fortreating the carbohydrate metabolism disturbance-related diseases suchas type II diabetes, insensitivity to insulin or obesity, etc.